Peptic ulcer disease and upper GI irritation are a significant burden in the US healthcare system and worldwide, caused in large part by the upper GI toxicity of ingested non-steroidal anti-inflammatory drugs (NSAIDs) and the pathogen H. pylori. The rationale for our work is that improved or alternative therapeutic strategies can be based on understanding the mechanisms of gastric defense rallied against such challenges. Preliminary studies show an essential role for both intracellular and extracellular Ca2+ mobilization in gastric repair of the surface epithelium. We hypothesize that this Ca2+ mobilization is a central regulator of repair that underlies the effects of diverse agonists shown to modulate the repair process, and that this pathway can be disrupted by NSAIDs and H. pylori. The objective of this application is to use the newly identified Ca2+ signals as heralds to identif the underlying upstream and downstream mechanisms mediating gastric repair, and as a tool to investigate if NSAIDs and/or H. pylori compromise repair via these pathways. We have pioneered optical technologies that allow real-time creation of focal damage and continual quantification of repair. Our work focusing on the repair of focal lesions in vivo will be extrapolated to experiments evaluating ulceration to enhance relevance to gastric pathologies observed clinically, and to study of gastric organoids as a potentially powerful new model of gastric epithelial function. Our first aim is to identify the initiating extracellular signals in damaged tissue that stimulate Ca2+ mobilization and gastric repair. We will focus on TFF2 and prostaglandins, shown to be essential to gastric repair, but whose role in Ca2+ signaling is untested. The second aim establishes the hierarchy of Ca2+-dependent intracellular signaling pathways that are essential to allow repair. Experiments are based on preliminary findings that show phospholipase C, and protein kinase C are required for efficient gastric repair in the intact stomach. Our third aim evaluates the downstream targets of Ca2+- dependent signaling that perform the work of gastric repair. We will evaluate the role of the NHE2 Na/H exchanger, shown to be essential for gastric repair as a downstream effector of TFF2. We will also evaluate Ca2+ -dependent regulation of the actin cytoskeleton, tight junctions, and the small GTPases that regulate these cellular structures during repair. All aims focus on the role of the cyclooxygenase (COX)/prostaglandin pathway inhibited by NSAIDs, and the first and third aims additionally examine changes in the studied pathways caused by infection with H. pylori. The outcomes will provide a unique window into understanding early factors in gastric pathogenesis, which have potential to lead to new targets and new strategies for interventions that can minimize gastric damage and speed healing.